ARTICLE
`
`doi:10.1006/mthe.2000.0116, available online at http://www.idealibrary.com on IDEAL
`
`VSV-G Pseudotyped Lentiviral Vector Particles Produced
`in Human Cells Are Inactivated by Human Serum
`Nicholas J. DePolo,* Joyce D. Reed,* Philip L. Sheridan,* Kay Townsend,* Sybille L. Sauter,*
`Douglas J. Jolly,* and Thomas W. Dubensky, Jr.*,1
`
`Chiron Corporation, Emeryville, California 94608
`
`Received for publication April 3, 2000, and accepted in revised form July 18, 2000
`
`Lentiviral vectors transduce dividing and postmitotic cells and thus are being developed toward
`therapies for many diseases affecting diverse tissues. One essential requirement for efficacy will
`be that vector particles are resistant to inactivation by human serum complement. Most animal
`studies with lentiviral vectors have utilized VSV-G pseudotyped envelopes. Here we demonstrate
`that VSV-G pseudotyped HIV and FIV vectors produced in human cells are inactivated by human
`serum complement, suggesting that alternative envelopes may be required for therapeutic effi-
`cacy for many clinical applications of lentiviral vectors.
`
`Key Words: lentivirus vector; retroviral vector; complement; resistance; human sera inactivation;
`VSV G pseudotyping.
`
`INTRODUCTION
`
`Two important recent advances in retroviral vector tech-
`nology are (i) the development of stable human packag-
`ing cell lines (PCLs) for production of Moloney murine
`leukemia virus (MLV) vectors that are resistant to inacti-
`vation by human serum complement (1–4) and (ii) the
`development of lentiviral vectors capable of transducing
`both dividing and postmitotic cells (5–12). To date,
`lentiviral vectors derived from human immunodeficien-
`cy virus (HIV) and feline immunodeficiency virus (FIV)
`have been produced by transient transfection or by
`induction of stable PCLs. These vectors are typically pro-
`duced in derivatives of the human 293 cell line and are
`predominantly pseudotyped with glycoprotein G from
`vesicular stomatitis virus (VSV-G) (5, 10). In contrast to
`MLV-based vectors, HIV and FIV VSV-G pseudotyped vec-
`tors have demonstrated good transduction efficiency in
`postmitotic cells of many tissues in animals, including
`retina, respiratory epithelium, muscle, brain, and liver
`(11, 13–15). VSV-G pseudotyped retroviral vectors are
`more uniformly infectious over a broad range of tissues
`and species, compared to vector particles containing the
`amphotropic or xenotropic envelopes (16, 17). Retroviral
`vectors produced by transient transfection methods are
`generally higher in titer when using VSV-G rather than
`alternative (e.g., amphotropic) envelopes, and VSV-G
`
`1To whom correspondence should be addressed at Chiron
`Corporation, 4560 Horton Street, Emeryville, CA 94608. Fax: (510) 923-
`2586. E-mail: tom_dubensky@cc.chiron.com.
`
`218
`
`also provides purification advantages due to the
`increased stability of the vector particle (16).
`Work by us and by others demonstrated that MLV-
`based retroviral vectors produced in certain human cells
`are resistant to inactivation by human complement (1–4,
`18). Testing of amphotropic MLV vector produced in
`human HT1080 cells showed that resistance to inactiva-
`tion by human serum in vitro correlated with highly
`increased in vivo systemic stability in chimpanzees fol-
`lowing intravenous administration. In contrast, MLV
`vectors produced in canine cells were rapidly susceptible
`to inactivation both in vitro and in vivo (2).
`Generally, complement resistance correlates with
`retroviral vectors produced in cells lacking (α1-3)galacto-
`syltransferase (αGT) activity, particularly certain human
`cells (19, 20). Complement directed by antibody specific
`for galactosyl (α1-3)galactosyl (αGal) terminal glycosidic
`epitopes, synthesized by αGT, plays an integral role in
`retroviral vector inactivation (20, 21). Other factors
`besides the producer cell, particularly the envelope gly-
`coprotein, also contribute in determining complement
`sensitivity (18, 22).
`MLV-based amphotropic vectors resistant to inactiva-
`tion by complement have been an important compo-
`nent in our development of a Factor VIII gene therapeu-
`tic for hemophilia A, which is administered intravenous-
`ly (23). Because lentiviral vectors may become an impor-
`tant vector for this clinical application as well as for oth-
`ers, we decided to determine the relative complement
`sensitivity of two primary lentiviral vectors, HIV and FIV,
`produced in human cell lines with either VSV-G or
`
`MOLECULAR THERAPY Vol. 2, No. 3, September 2000
`Copyright 䊚 The American Society of Gene Therapy
`1525-0016/00 $35.00
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`Page 1 of 5
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`KELONIA EXHIBIT 1020
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`amphotropic envelopes. Defining the parameters which
`result in complement resistance is an important step
`toward developing lentiviral vectors that are broadly use-
`ful for human gene therapy applications.
`
`MATERIALS AND METHODS
`
`Cells. BHK-21, HT1080, and 293T cells were maintained in DMEM
`(Gibco-BRL) containing 10% fetal bovine serum (FBS).
`
`Vectors. All vectors (MLV, HIV, and FIV) were produced by parallel
`methods through transfection of human 293T cells. Cotransfections of
`vector, envelope, and gag-pol plasmids were performed as described pre-
`viously (7, 9, 16). Crude titers ranged between 1 ⫻ 105 and 5 ⫻ 106 blue
`colony-forming units per ml (BCFU/ml) in various preparations, with
`VSV-G preparations averaging 1–2 ⫻ 106 BCFU/ml and amphotropic
`enveloped preparations about 1–3 ⫻ 105 BCFU/ml. To standardize input
`titers and preparation purity, all VSV-G and amphotropic envelope vec-
`tors encoding β-galactosidase were purified and concentrated by low-
`speed centrifugation (24) before resuspension and dilution in growth
`medium to equivalent titer [106 BCFU/ml]. Aliquots were frozen at
`−70⬚C, for later use in assays for stability in 80% human sera, for both G
`and amphotropic envelope preparations of all vectors. Titer recovery was
`typically in the 50–90% range, and control tests of crude versus concen-
`trated vector showed very similar serum sensitivity.
`
`Titer assays. Vector samples from in vitro or in vivo assays were titered
`on HT080 target cells on six-well plates in the presence of 8 µg/ml poly-
`brene (Sigma). BCFU titers were determined following X-gal staining fol-
`lowing standard methods, as described previously (2). Determination of
`VSV titer was by plaque assay of individual serial dilutions of samples
`(25).
`
`In vitro serum inactivation assays. Human sera used were either (A)
`pooled complement-active normal human sera (approximately five or
`more individuals per pool; Quidel, San Diego, CA) or (B) normal human
`sera from four individuals. Individual human sera were prepared and
`tested to verify normal complement activity, as described previously (2).
`Equal input titers of each virus or vector type were used in an experi-
`ment. To determine serum inactivation, either viral vector or VSV was
`diluted fivefold in normal human sera (NHS), heat-inactivated sera (HIS;
`incubated 1 h at 56⬚C), or control fetal bovine sera. All incubations were
`for 1 h at 37⬚C in 80% test sera with 100- to 200-µl reaction volumes. Sera
`absorption experiments were performed essentially as described by Beebe
`et al. (26). Briefly, BHK-21 cells (or HT1080 cells which gave similar
`results) were plated 24 h previously at 1 ⫻ 107 cells per T-75 flask and
`infected or mock infected with VSV-Indiana at an m.o.i. of 10 in 1 ml of
`medium for 1 h at 37⬚C and rinsed and fresh medium was added. After 4
`h incubation, trypsinized cells were rinsed 2⫻ with growth medium and
`2⫻ with PBS and then incubated on ice for 6 h with 0.5 ml of pooled
`normal human sera, Quidel Lot 2. Cells and debris were removed by two
`centrifugations for 0.5 h at 15,000g in a microfuge at 4⬚C. Residual VSV
`was inactivated by UV (30 s in a Stratagene (San Diego, CA) UV Cross-
`linker, and sera were stored on ice until use in assays.
`
`RESULTS AND DISCUSSION
`
`VSV is sensitive to inactivation by human serum. To deter-
`mine the serum sensitivity of various VSV-G pseudo-
`typed vectors, it is logical to first measure the sensitivity
`of VSV grown in nonhuman or human cells, which has
`been explored by several groups. Beebe et al. reported
`that VSV was equally sensitive when grown in BHK or
`certain human cells (26), and Welsh et al. observed that
`VSV was equally sensitive to human serum when grown
`in either αGal(+) or αGal(−) human cells (27), supporting
`these findings. Conversely, Thiry et al. (28) and Takeuchi
`
`ARTICLE
`
`et al. (29) found that human cell-propagated VSV had
`reduced sensitivity to human sera. In these reports, sera
`assay concentrations ranged from 10 to 50% and this
`along with cell line and other assay variations may
`explain some differences in findings. We have estab-
`lished a standardized higher percentage serum assay that
`has predictive value for in vivo circulation stability in pri-
`mates, as we have described previously (2). Thus, we first
`assessed the stability of VSV propagated in BHK-21 ham-
`ster or HT1080 human cell lines with 80% serum assays
`in multiple individuals and two pooled sera lots.
`HT1080-propagated VSV was substantially inactivated,
`100- to 400-fold, while the BHK-21 cell-propagated virus
`was more strongly inactivated, ⬎10,000-fold (data not
`shown). These results are in general agreement with the
`results of Takeuchi et al. (29) with VSV propagated in
`these two cell lines, confirming that VSV grown in
`human cells retains substantial sensitivity to inactivation
`by human sera.
`VSV-G pseudotyped oncoretroviral and lentiviral vectors
`are sensitive to inactivation by human serum. We compared
`the relative resistance to human serum inactivation of
`matched titer, human 293 cell-produced MLV, and
`lentiviral (HIV and FIV) vectors containing either VSV-G
`or amphotropic (4070A) envelope, to determine whether
`the results observed with VSV also correlated with VSV-G
`pseudotyped vector serum sensitivity. MLV, HIV, and FIV
`vectors containing the amphotropic envelope were
`resistant to inactivation by human serum, while the cor-
`responding VSV-G pseudotyped vectors were nearly com-
`pletely inactivated by the same experimental conditions
`(Fig. 1). The relative stability of all vectors observed in
`HIS suggests that the majority of inactivation in this lot
`of pooled sera was due to a heat-labile component, pre-
`sumably complement. We have shown previously with
`similar matched titer (~2 ⫻ 105 BCFU/ml), high serum
`percentage assays that the level of in vitro human serum
`sensitivity correlated with decreased circulation half-life
`in primates phylogenetically close to human (2). Thus,
`these results suggest strongly that VSV-G pseudotyped
`lentiviral vectors might not be efficacious for human
`gene therapy applications requiring systemic administra-
`tion.
`The profile of oncoretroviral and lentiviral vector
`human serum sensitivity reported here is more striking
`than previous publications which suggested that VSV or
`VSV-G pseudotyped MLV or HIV vectors produced in
`αGal-negative cells were substantially more resistant to
`serum inactivation (29, 30). Several possibilities may
`explain why we found greater sensitivity to serum,
`including (i) we compared 80% serum inactivation
`between vectors all produced by equivalent methods, (ii)
`we tested at matched low titers to avoid saturation effects
`(2), and (iii) we evaluated inactivation in multiple sera
`samples. It seems likely that high percentage sera assays
`may be more relevant to certain in vivo administration
`routes, such as intravenous. With all these sera, VSV-G
`pseudotyped MLV, HIV, and FIV vectors produced in
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`MOLECULAR THERAPY Vol. 2, No. 3, September 2000
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`ARTICLE
`
`FIG. 1. VSV-G pseudotyped lentiviral and murine retroviral vectors produced in human cells are inactivated by human serum. Inactivation in pooled human
`sera of MLV, HIV, or FIV vectors containing the VSV-G or amphotropic envelope was measured by titer assays performed on HT1080 cells and staining with X-
`gal (BCFU/ml) (8). Survival percentage indicates the fraction of titer remaining in the NHS (normal human sera, Quidel pool, Lot 1) or HIS (heat-inactivated
`sera) versus the FBS control. All assays were repeated a minimum of two times, in triplicate, and mean values (±SE) from representative experiments are shown.
`A, B, and C represent relative inactivation of MLV, HIV, and FIV vectors, respectively.
`
`293T cells by transient methods were substantially sensi-
`tive to serum inactivation. Most importantly, previous
`studies have not tested matched amphotropic vectors in
`parallel assays, and the results here indicate their com-
`paratively greater serum stability, even under these strin-
`gent conditions.
`VSV-G-specific antibodies mediate human 293T cell pro-
`duced G-vector inactivation. Although the results in Fig. 1
`reveal relatively little inactivation of G pseudotyped vec-
`tor in HI sera from a specific lot of pooled human sera,
`certain human individual or pooled lots of sera resulted
`in more heat-stabile (i.e., noncomplement) inactivation.
`This result was specific to G vectors, as shown in Table 1.
`Pooled sera Lot 2 showed the highest heat-stabile G-vec-
`tor inactivation, while a third lot tested was more simi-
`lar to Lot 1. These results with different pooled human
`serum lots suggest variable levels of innate neutralizing
`antibody to VSV-G in human sera. This is similar to the
`VSV-G innate antibodies in certain mouse strains recent-
`ly described by Ochsenbein et al., which were low level,
`but shown to have potent effects on injected virus in
`their studies (31). As heat-labile inactivation may also be
`mediated by complement-fixing antibodies (26), it was
`of interest to determine whether antibodies specific to
`VSV-G could be preabsorbed. These sera were absorbed
`with either mock infected or 4-h VSV-infected BHK cells
`
`(expressing VSV-G on their surface) as described under
`Materials and Methods. The native or absorbed sera were
`tested against either human 293T-produced VSV-G MLV
`vector or canine D17-produced amphotropic vector (2),
`as a control for complement activity. The mock-infected
`BHK absorption caused only a partial reduction in sera
`inactivation to a similar extent for both G vector and
`control vector. In contrast, the absorption with VSV-BHK
`cells nearly eliminated both heat-stabile and heat-labile
`sera inactivation of the G vector, but not the control.
`These results suggest that absorbable factors, such as G-
`specific antibodies, mediate human 293T cell produced
`G-vector inactivation, both directly and by complement
`activation.
`Because human cells, such as 293, are αGal-negative,
`it is unlikely that antibodies to this epitope play a role in
`serum inactivation of these G pseudotyped vectors.
`Although it is well established that αGal glycosylation
`epitopes are a primary determinant for targeting
`oncoretroviral and lentiviral vector inactivation in
`human sera, it is also apparent that not all mechanisms
`for this process are fully understood. Complement-resist-
`ant retroviral vectors can be produced in nonhuman,
`nonprimate mink and ferret brain cells, both of which
`are αGal-positive (18, 22). Furthermore, there is a broad
`spectrum of relative stability in human sera of vectors
`
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`MOLECULAR THERAPY Vol. 2, No. 3, September 2000
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`TABLE 1
`Amphotropic and VSV-G MLV Vector Sensitivity to
`Sera Pre- and Postabsorptions
`Survival (%)a
`Human sera tested
`D17 ampho ␤-gal
`293T VSV-G ␤-gal
`
`HIS
`NHS
`HIS-BHK absorbedb
`NHS-BHK absorbed
`HIS-VSV/BHK
`absorbed
`NHS-VSV/BHK
`absorbed
`
`61 ⫾ 17
`0.045 ⫾ 0.02
`74 ⫾ 21
`0.35 ⫾ 0.15
`47 ⫾ 12
`
`0.16 ⫾ 0.08
`
`0.04 ⫾ 0.02
`⭐ 0.01*
`0.67 ⫾ 0.29
`⭐ 0.01*
`129⫾ 30
`
`39 ⫾ 8
`
` a Survival percentage ⫾ SE is the fraction of vector titer remaining after incubation in
`test sera (HIS, heat inactivated sera or HS, normal human sera) relative to the FBS control
`incubation.
` b Preabsorption conditions for the Quidel Lot 2 pooled sera are described in the text. *
`indicates samples with no detectable titer observed. Representative results are shown
`from one of two experiments.
`
`produced in a variety of different human cell lines, all of
`which are apparently αGal-negative (18, 32). Interaction
`between vector envelope and producer cell membrane
`components may also play an interdependent role in
`conferring complement resistance of the vector (22).
`Thus, although following certain guidelines such as pro-
`duction in human cells like 293 can help produce vectors
`that are more stable in human sera, there still remain
`many other factors that determine complement resist-
`ance.
`The findings reported here suggest that VSV-G
`pseudotyping-conferred complement sensitivity is not
`primarily due to VSV-G specific neutralizing antibody,
`since only certain HIS samples tested significantly neu-
`tralize vector. Our sera VSV G-Ab absorption experiments
`support the proposal from early VSV experiments that
`VSV-G specific nonneutralizing IgM class antibodies may
`bind to a highly conserved epitope(s) and direct classical
`pathway complement inactivation (26, 28). However,
`despite repeated efforts, we have so far been unable to
`generate VSV “antibody-escape mutants” that were able
`to propagate in human serum (data not shown). These
`results suggest that there may be critical epitope(s) that
`are essential for viral function.
`Other mechanisms of VSV-G vector inactivation
`could be involved; for example, VSV-G may interact dif-
`ferently with human cell membrane complement con-
`trol proteins, like CD-55 or CD-59 (33). VSV-G might
`either restrict vector membrane incorporation or block
`function of these cell proteins. According to this mecha-
`nism, VSV-G would prevent one part of multiple syner-
`gistic complement resistance mechanisms. This notion is
`supported by the observation that HIV and other
`enveloped viruses incorporate species-specific membrane
`complement control proteins (34–37). Whatever the
`mechanism or combination of mechanisms for inactiva-
`tion may be, VSV-G pseudotyped lentiviral vectors pro-
`
`ARTICLE
`
`duced in human cells are inactivated by human serum.
`In summary, we have demonstrated that lentiviral
`vectors containing an amphotropic envelope may have
`substantial advantages compared to VSV-G pseudotyped
`vectors for certain human gene transfer applications, par-
`ticularly those requiring intravenous administration.
`Although the extent of inactivation of VSV-G pseudo-
`typed vectors across several pooled and individual
`human sera was somewhat variable, the level of suscepti-
`bility was always substantial. This observation has impor-
`tant implications for efficacy in vivo, as well as for the
`design and creation of new lentiviral production and PCL
`systems.
`
`ACKNOWLEDGMENTS
`
`We thank the many scientists at Chiron who contributed to the production of
`vectors and cell reagents. In particular, we thank M. Gasmi, J. Glynn, and B.
`Belli for providing HIV vector plasmids, helpful discussions, and critical read-
`ing.
`
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`MOLECULAR THERAPY Vol. 2, No. 3, September 2000
`Copyright 䊚 The American Society of Gene Therapy
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